Browsing by Subject "REMAP"

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  • Tanhuanpää, Pirjo; Erkkilä, Maria; Kalendar, Ruslan; Schulman, Alan Howard; Manninen, Outi (2016)
    Timothy (Phleum pratense L.), a cool-season hexaploid perennial, is the most important forage grass species in Nordic countries. Earlier analyses of genetic diversity in a collection of 96 genebank accessions of timothy with SSR markers demonstrated high levels of diversity but could not resolve population structure. Therefore, we examined a subset of 51 accessions with REMAP markers, which are based on retrotransposons, and compared the diversity results with those obtained with SSR markers.
  • Li, Shitian; Ramakrishnan, Muthusamy; Vinod, K. K.; Kalendar, Ruslan; Yrjälä, Kim; Zhou, Mingbing (2020)
    Bamboo, a non-timber grass species, known for exceptionally fast growth, is a commercially viable crop. Long terminal repeat (LTR) retrotransposons, the main class I mobile genetic elements in plant genomes, are highly abundant (46%) in bamboo contributing to genome diversity. They play significant roles in the regulation of gene expression, chromosome size and structure as well as in genome integrity. Inter-retrotransposon amplified polymorphism (IRAP) is a high-throughput method to study the genetic diversity of plant species. Till date, there are no markers based on Transposable Elements (TEs) for the bamboo genome and no reports on bamboo genetic diversity using the IRAP method. Phyllostachys is an Asian bamboo, the largest group in the bamboo subfamily, Bambusoideae, and it is of great economic value due to its fast growth. The structure of LTR-retrotransposon superfamilies, Ty3-gypsy and Ty1-copia, were analysed in the bamboo genome using LTRharvest and LTRdigest software. A total of 98,850 LTR retrotransposons with both ends of intact LTR sequences were identified, grouped into 64,281 clusters/scaffolds, using CD-HIT software. Among the total of 64,281 clusters, 13 clusters had more than 30 copy numbers of LTR sequences and at least one copy had all intact protein domains such as gag protein and polyprotein. Based on the high copy numbers of conserved LTR sequences, a total of 16 IRAP primers were developed. All these IRAP primers were used to study the genetic diversity and population structure of the Asian bamboo. AMOVA analysis was done for 58 Asian bamboo species collected from nine different provinces of China, from Italy and America. In the bamboo species, these IRAP primers produced a total of 3340 amplicons with an average of 98% polymorphism. The 58 Asian bamboo species were grouped into two major clusters and four sub-clusters, based on UPGMA analysis. UPGMA cluster analysis was corroborated by statistical analyses of genetic similarity coefficients. Structure analysis showed that the bamboo species could be divided into four subpopulations (K = 4): SP1, SP2, SP3 and SP4. All SPs had an admixture of alleles. AMOVA analysis showed that higher genetic variations occurred within populations (75%) rather than among populations (25%). The study highlights the usability of IRAP in Asian bamboo to determine inter-species variability using retrotransposon markers.
  • Khapilina, Oxana; Raiser, Olesya; Danilova, Alevtina; Shevtsov, Vladislav; Turzhanova, Ainur; Kalendar, Ruslan (2021)
    Analysis of the genetic diversity of natural populations of threatened and endangered species of plants is a main aspect of conservation strategy. The endangered species Allium altaicum is a relict plant of the Ice Age and natural populations are located in extreme climatic conditions of Kazakstan's Altai Mountains. Mobile genetic elements and other interspersed repeats are basic components of a eukaryote genome, which can activate under stress conditions and indirectly promote the survival of an organism against environmental stresses. Detections of chromosomal changes related to recombination processes of mobile genetic elements are performed by various PCR methods. These methods are based on interspersed repeat sequences and are an effective tool for research of biological diversity of plants and their variability. In our research, we used conservative sequences of tRNA primer binding sites (PBS) when initializing the retrotransposon replication as PCR primers to research the genetic diversity of 12 natural populations of A. altaicum found in various ecogeographic conditions of the Kazakhstani Altai. High efficiency of the PBS amplification method used was observed already at the intrapopulation level. Unique amplicons representative of a certain population were found at the intrapopulation level. Analysis of molecular dispersion revealed that the biodiversity of populations of mountainous and lowland A. altaicum is due to intrapopulation differences for climatic zones of habitation. This is likely conditional upon predominance of vegetative reproduction over seed reproduction in some populations. In the case of vegetative reproduction, somatic recombination related to the activity of mobile genetic elements are preserved in subsequent generations. This leads to an increase of intrapopulation genetic diversity. Thus, high genetic diversity was observed in populations such as A. altaicum located in the territory of the Kalbinskii Altai, whereas the minimum diversity was observed in the populations of the Leninororsk ecogeographic group. Distinctions between these populations were also identified depending on the areas of their distribution. Low-land and mid-mountain living environments are characterized by a great variety of shapes and plasticity. This work allowed us to obtain new genetic data on the structure of A. altaicum populations on the territory of the Kazakhstan Altai for the subsequent development of preservation and reproduction strategies for this relict species.
  • Turzhanova, Ainur; Khapilina, Oxana; Tumenbayeva, A; Shevtsov, Vladislav; Raiser, Olesya; Kalendar, Ruslan (2020)
    The genus Alternaria is a widely distributed major plant pathogen that can act as a saprophyte in plant debris. Fungi of this genus frequently infect cereal crops and cause such diseases as black point and wheat leaf blight, which decrease the yield and quality of cereal products. A total of 25 Alternaria sp. isolates were collected from germ grains of various wheat cultivars from different geographic regions in Kazakhstan. We investigated the genetic relationships of the main Alternaria species related to black point disease of wheat in Kazakhstan, using the inter-primer binding site (iPBS) DNA profiling technique. We used 25 retrotransposon-based iPBS primers to identify the differences among and within Alternaria species populations, and analyzed the variation using clustering (UPGMA) and statistical approaches (AMOVA). Isolates of Alternaria species clustered into two main genetic groups, with species of A.alternata and A.tennuissima forming one cluster, and isolates of A. infectoria forming another. The genetic diversity found using retrotransposon profiles was strongly correlated with geographic data. Overall, the iPBS fingerprinting technique is highly informative and useful for the evaluation of genetic diversity and relationships of Alternaria species.
  • Vasilyeva, Yulia; Chertov, Nikita; Nechaeva, Yulia; Sboeva, Yana; Pystogova, Nina; Boronnikova, Svetlana; Kalendar, Ruslan (2021)
    In order to carry out activities aimed at conservation and rational use of forest resources; it is necessary to study the main forest-forming plant species in detail. Scots pine (Pinus sylvestris L., Pinaceae) is mainly found in the boreal forests of Eurasia and is not so often encountered in the east of the East European Plain. The aim of the study was to study the genetic diversity, structure and differentiation of Scots pine populations in the east of the East European Plain. We studied ten populations of P. sylvestris using the Inter Simple Sequence Repeats (ISSR)-based DNA polymorphism detection method. Natural populations are demonstrated by relatively high rates of genetic diversity (He = 0.167; ne = 1.279; I = 0.253). At the same time, there is a tendency for a decrease in the genetic diversity of the studied populations of P. sylvestris from west to east. Analysis of the genetic structure shows that the studied populations are highly differentiated (GST = 0.439), the intrapopulation component accounts for about 56% of the genetic diversity. Using various algorithms for determining the spatial genetic structure, it is found that the studied populations form two groups of populations in accordance with geographic location. With the help of a genetic originality coefficient, populations with specific and typical gene pools are identified. They are recommended as sources of genetic diversity and reserves for the conservation of genetic resources of the species
  • Ghonaim, Marwa; Kalendar, Ruslan; Barakat, Hoda; Elsherif, Nahla; Ashry, Naglaa; Schulman, Alan (2020)
    Maize is one of the world’s most important crops and a model for grass genome research. Long terminal repeat (LTR) retrotransposons comprise most of the maize genome; their ability to produce new copies makes them efficient high-throughput genetic markers. Inter-Retrotransposon-Amplified Polymorphisms (IRAPs) were used to study the genetic diversity of maize germplasm. Five LTR retrotransposons (Huck, Tekay, Opie, Ji, and Grande) were chosen, based on their large number of copies in the maize genome, whereas polymerase chain reaction primers were designed based on consensus LTR sequences. The LTR primers showed high quality and reproducible DNA fingerprints, with a total of 677 bands including 392 polymorphic bands showing 58% polymorphism between maize hybrid lines. These markers were used to identify genetic similarities among all lines of maize. Analysis of genetic similarity was carried out based on polymorphic amplicon profiles and genetic similarity phylogeny analysis. This diversity was expected to display ecogeographical patterns of variation and local adaptation. The clustering method showed that the varieties were grouped into three clusters differing in ecogeographical origin. Each of these clusters comprised divergent hybrids with convergent characters. The clusters reflected the differences among maize hybrids and were in accordance with their pedigree. The IRAP technique is an efficient high-throughput genetic marker-generating method.
  • Kalendar, Ruslan; Shustov, Alexandr; Schulman, Alan (2021)
    Genome walking (GW), a strategy for capturing previously unsequenced DNA fragments that exist in proximity to a known sequence tag, is currently predominantly based on PCR. Recently developed PCR-based methods allow for combining of sequence-specific primers with designed capturing primers capable of annealing to unknown DNA targets, which offer the rapidity and effectiveness of PCR. This study presents a methodological improvement to the previously described GW technique known as Palindromic Sequence-Targeted PCR (PST-PCR). Like PST-PCR, this new method (called PST-PCR v.2) relies on targeting of capturing primers to palindromic sequences arbitrarily present in natural DNA templates. PST-PCR v.2 consists of two rounds of PCR. The first round uses a combination of one sequence-specific primer with one capturing (PST) primer. The second round uses a combination of a single (preferred) or two universal primers; one anneals to a 5’ tail attached to the sequence-specific primer and the other anneals to a different 5’ tail attached to the PST primer. The key advantage of PST-PCR v.2 is the convenience of using a single universal primer with invariable sequences in GW processes involving various templates. The entire procedure takes approximately 2–3 hours to produce the amplified PCR fragment, which contains a portion of a template flanked by the sequence-specific and capturing primers. PST-PCR v.2 is highly suitable for simultaneous work with multiple samples. For this reason, PST-PCR v.2 can be applied beyond the classical task of GW for studies in population genetics, in which PST-PCR v.2 is a preferred alternative to amplified fragment length polymorphism (AFLP) or next-generation sequencing. Furthermore, the conditions for PST-PCR v.2 are easier to optimize, as only one sequence-specific primer is used. This reduces non-specific Random Amplified Polymorphic DNA (RAPD)-like amplification and formation of non-templated amplification. Importantly, akin to the previous version, PST-PCR v.2 is not sensitive to template DNA sequence complexity or quality. This study illustrates the utility of PST-PCR v.2 for transposon display, which is a method to characterize inter- or intra-specific variability related to transposon integration sites. The Ac transposon sequence in the corn (Zea mays) genome was used as a sequence tag during the transposon display procedure to characterize the Ac integration sites.
  • Vuorinen, Anssi L.; Kalendar, Ruslan; Fahima, Tzion; Korpelainen, Helena; Nevo, Eviatar; Schulman, Alan H. (2018)
    Wild emmer wheat (Triticum turgidum ssp. dicoccoides) is the wild ancestor of all cultivated tetraploid and hexaploid wheats and harbors a large amount of genetic diversity. This diversity is expected to display eco-geographical patterns of variation, conflating gene flow, and local adaptation. As self-replicating entities comprising the bulk of genomic DNA in wheat, retrotransposons are expected to create predominantly neutral variation via their propagation. Here, we have examined the genetic diversity of 1 Turkish and 14 Israeli populations of wild emmer wheat, based on the retrotransposon marker methods IRAP and REMAP. The level of genetic diversity we detected was in agreement with previous studies that were performed with a variety of marker systems assaying genes and other genomic components. The genetic distances failed to correlate with the geographical distances, suggesting local selection on geographically widespread haplotypes (‘weak selection’). However, the proportion of polymorphic loci correlated with the population latitude, which may reflect the temperature and water availability cline. Genetic diversity correlated with longitude, the east being more montane. Principal component analyses on the marker data separated most of the populations.